Background: Viral load (VL) determination in patients with human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV) and hepatitis C virus (HCV) is essential for proper patient... Show moreBackground: Viral load (VL) determination in patients with human immunodeficiency virus type 1 (HIV-1), hepatitis B virus (HBV) and hepatitis C virus (HCV) is essential for proper patient management and follow-up. New molecular platforms have been developed to fully automate these diagnostic assays.Objective: Evaluation of the clinical performance of HIV-1, HBV and HCV VL assays on the Alinity m (Abbott) and NeuMoDx (Qiagen) molecular platforms.Method: Test panels of the three viruses have been compiled of 100 plasma and/or serum samples per target containing non-detectable, non-quantifiable and quantifiable VLs. All samples were retrospectively tested on the Alinity m and NeuMoDx platforms according to manufacturers' instructions. Results: A total of 74, 86 and 66 samples with valid results for both platforms were included in the HIV-1, HBV and HCV analysis respectively. Overall qualitative agreement of the assays on both platforms was 78% for HIV-1, 93% for HBV and 100% for HCV. Quantitative agreement (less than 0.5 log difference) was shown to be 68% for HIV-1, 68% for HBV and 94% for HCV.Conclusion: The Alinity m and NeuMoDx HCV assay have a comparable performance. Quantification differences in the HIV-1 assay were mostly apparent in the lower VLs and under-quantification of the NeuMoDx HBV assay was observed. Show less
Background: The detection and follow up of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) viral loads (VL) are crucial in the management of immunocompromised patients. Recently, molecular CE... Show moreBackground: The detection and follow up of cytomegalovirus (CMV) and Epstein-Barr virus (EBV) viral loads (VL) are crucial in the management of immunocompromised patients. Recently, molecular CE-IVD assays for detection and quantification of CMV and EBV have been launched for use on the random-access and sample-to-result NeuMoDx 96 and 288 platforms (Qiagen). Objective: Evaluating the qualitative and quantitative performance of the NeuMoDx CMV and EBV assays in clinical specimens compared to a lab developed tests (LDT) and the CE-IVD assays on the Abbott m2000 system. Method: Both a prospective and a retrospective panel, compiled of non-detectable (ND), non-quantifiable (NQ) and quantifiable VLs in plasma samples have been evaluated for both CMV and EBV: NeuMoDx versus LDT and NeuMoDx versus Abbott m2000. Quantitative agreement was determined for samples with a quantifiable VL on both systems. Results: Qualitative and quantitative agreement between the NeuMoDx and LUMC's LDT CMV assays was 88%. Qualitative agreement between the NeuMoDx and Abbott m2000 CMV assays was 92% and quantitative agreement was 87%. Qualitative and quantitative agreement between the NeuMoDx and the LDT EBV assays was 87%. Qualitative agreement between the NeuMoDx and Abbott m2000 EBV assays was 91% and quantitative agreement was 0%. Conclusion: These data show that the NeuMoDx assays are suitable for both detection and quantification of CMV and EBV in a medium-to high throughput diagnostic setting, but that differences in sensitivity and quantification (for EBV, NeuMoDx versus Abbott m2000) warrant an extensive transition period when using the respective assays for following VL in patient samples. Show less
Introduction: Human polyomaviruses (HPyVs) cause disease in immunocompromised patients. BK polyomavirus (BKPyV) for instance persistently infects the kidneys. In kidney transplant recipients, (KTRs... Show moreIntroduction: Human polyomaviruses (HPyVs) cause disease in immunocompromised patients. BK polyomavirus (BKPyV) for instance persistently infects the kidneys. In kidney transplant recipients, (KTRs) BKPyV can cause allograft nephropathy. JCPyV, MCPyV, TSPyV and HPyV9 reside in the kidneys too, or have been detected in urine. In this study, we investigate exposure to JCPyV, MCPyV, TSPyV and HPyV9 after kidney transplantation by serological means.Materials and methods: Serum samples from 310 KTR collected before and 6 months after transplantation (n = 620), from 279 corresponding kidney donors collected before transplantation, and from blood donor controls collected one year apart (n = 174) were assessed for HPyV species-specific IgG responses using a multiplex immunoassay. KTR HPyV IgG kinetics were compared to those of healthy blood donors by linear mixed modeling, and related to those of their donors by linear regression.Results: In the KTR, increased IgG levels during follow-up were observed for JCPyV (14.8%), MCPyV (7.1%), TSPyV (10.6%), and for HPyV9 (8.1%), while blood donor antibody levels remained stable. Seroconversion was observed for JCPyV (6.5%), MCPyV (2.3%), TSPyV (1.3%), and for HPyV9 (6.5%). The linear mixed model analysis showed that antibody increase was significant for JCPyV (p < 0.001) and HPyV9 (p < 0.001). Post transplant JCPyV and HPyV9 antibody responses were associated with donor antibody levels against these HPyVs, respectively.Conclusions: KTR are exposed to JCPyV and HPyV9 after transplantation. Whether the allograft serves as the source, as indicated by the donor serostatus association, deserves further study. Show less
Background: Diagnosis of infections in returning international travellers can be challenging because of the broad spectrum of potential infectious etiologies potentially involved. Viral metagenomic... Show moreBackground: Diagnosis of infections in returning international travellers can be challenging because of the broad spectrum of potential infectious etiologies potentially involved. Viral metagenomic next-generation sequencing (mNGS) has the potential to detect any virus present in a patient sample and is increasingly being used for difficult to diagnose cases. The aim of this study was to analyze the performance of mNGS for viral pathogen detection in the clinical setting of international travellers returning with febrile illness. Methods: Thirty-eight serum samples from international travellers returning with febrile illness and presenting at the outpatient clinic of the Leiden University Medical Center in the Netherlands in the time period 2015-2016 were selected retrospectively. Samples were processed for viral metagenomic sequencing using a probe panel capturing all known vertebrate viruses. Bioinformatic analysis was performed using Genome Detective software for metagenomic virus detection. Metagenomic virus findings were compared with viral pathogen detection using conventional methods. Results: In 8 out of the 38 patients (21%), a pathogenic virus was detected by mNGS. All viral pathogens detected by conventional assays were also detected by mNGS: dengue virus (n=4 patients), Epstein-Barr virus (n=2), hepatitis B virus (n=1). In addition, mNGS resulted in additional pathogenic findings in 2 patients (5%): dengue virus (n=1), and hepatitis C virus (n=1). Non-pathogenic viruses detected were: GB virus C (n=1) and torque teno viruses (n=3). High genome coverage and depth using capture probes enabled typing of the dengue viruses detected. Conclusions: Viral metagenomics has the potential to assist the detection of viral pathogens and co-infections in one step in international travellers with a febrile syndrome. Furthermore, viral enrichment by probes resulted in high genome coverage and depth which enabled dengue virus typing. Show less
Introduction: Metagenomic sequencing is increasingly being used in clinical settings for difficult to diagnose cases. The performance of viral metagenomic protocols relies to a large extent on the... Show moreIntroduction: Metagenomic sequencing is increasingly being used in clinical settings for difficult to diagnose cases. The performance of viral metagenomic protocols relies to a large extent on the bioinformatic analysis. In this study, the European Society for Clinical Virology (ESCV) Network on NGS (ENNGS) initiated a benchmark of metagenomic pipelines currently used in clinical virological laboratories.Methods: Metagenomic datasets from 13 clinical samples from patients with encephalitis or viral respiratory infections characterized by PCR were selected. The datasets were analyzed with 13 different pipelines currently used in virological diagnostic laboratories of participating ENNGS members. The pipelines and classification tools were: Centrifuge, DAMIAN, DIAMOND, DNASTAR, FEVIR, Genome Detective, Jovian, MetaMIC, MetaMix,One Codex, RIEMS, VirMet, and Taxonomer. Performance, characteristics, clinical use, and user-friendliness of these pipelines were analyzed.Results: Overall, viral pathogens with high loads were detected by all the evaluated metagenomic pipelines. In contrast, lower abundance pathogens and mixed infections were only detected by 3/13 pipelines, namely DNASTAR, FEVIR, and MetaMix. Overall sensitivity ranged from 80% (10/13) to 100% (13/13 datasets). Overall positive predictive value ranged from 71-100%. The majority of the pipelines classified sequences based on nucleotide similarity (8/13), only a minority used amino acid similarity, and 6 of the 13 pipelines assembled sequences de novo. No clear differences in performance were detected that correlated with these classification approaches. Read counts of target viruses varied between the pipelines over a range of 2-3 log, indicating differences in limit of detection.Conclusion: A wide variety of viral metagenomic pipelines is currently used in the participating clinical diagnostic laboratories. Detection of low abundant viral pathogens and mixed infections remains a challenge, implicating the need for standardization and validation of metagenomic analysis for clinical diagnostic use. Future studies should address the selective effects due to the choice of different reference viral databases. Show less
The main challenge of immunosuppressive therapy after solid organ transplantation is to create a new immunological balance that prevents organ rejection and does not promote opportunistic infection... Show moreThe main challenge of immunosuppressive therapy after solid organ transplantation is to create a new immunological balance that prevents organ rejection and does not promote opportunistic infection. Torque teno virus (TTV), a ubiquitous and non-pathogenic single-stranded DNA virus, has been proposed as a marker of functional immunity in immunocompromised patients. Here we investigate whether TTV loads predict the risk of common viral infection and allograft rejection in kidney transplantation recipients.In a retrospective cohort of 389 kidney transplantation recipients, individual TTV loads in were measured by qPCR in consecutive plasma samples during one year follow-up. The endpoints were allograft rejection, BK polyomavirus (BKPyV) viremia and cytomegalovirus (CMV) viremia. Repeated TTV measurements and rejection and infection survival data were analysed in a joint model.During follow-up, TTV DNA detection in the transplant recipients increased from 85 to 100%. The median viral load increased to 107 genome copies/ml within three months after transplantation. Rejection, BKPyV viremia and CMV viremia occurred in 23%, 27% and 17% of the patients, respectively. With every 10-fold TTV load-increase, the risk of rejection decreased considerably (HR: 0.74, CI 95%: 0.71-0.76), while the risk of BKPyV and CMV viremia remained the same (HR: 1.03, CI 95%: 1.03-1.04 and HR: 1.01, CI 95%: 1.01-1.01).In conclusion, TTV load kinetics predict allograft rejection in kidney transplantation recipients, but not the BKPyV and CMV infection. The potential use of TTV load levels as a guide for optimal immunosuppressive drug dosage to prevent allograft rejection deserves further validation. Show less
Metagenomic next-generation sequencing (mNGS) is an untargeted technique for determination of microbial DNA/RNA sequences in a variety of sample types from patients with infectious syndromes. mNGS... Show moreMetagenomic next-generation sequencing (mNGS) is an untargeted technique for determination of microbial DNA/RNA sequences in a variety of sample types from patients with infectious syndromes. mNGS is still in its early stages of broader translation into clinical applications. To further support the development, implementation, optimization and standardization of mNGS procedures for virus diagnostics, the European Society for Clinical Virology (ESCV) Network on Next-Generation Sequencing (ENNGS) has been established. The aim of ENNGS is to bring together professionals involved in mNGS for viral diagnostics to share methodologies and experiences, and to develop application guidelines. Following the ENNGS publication Recommendations for the introduction of mNGS in clinical virology, part I: wet lab procedure in this journal, the current manuscript aims to provide practical recommendations for the bioinformatic analysis of mNGS data and reporting of results to clinicians. Show less
Background: Apart from major health concerns associated to the SARS-coronavirus-2 (SARS-CoV-2) pandemic, also the diagnostic workflow encountered serious problems. Limited availability of kit... Show moreBackground: Apart from major health concerns associated to the SARS-coronavirus-2 (SARS-CoV-2) pandemic, also the diagnostic workflow encountered serious problems. Limited availability of kit components, buffers and even plastics has resulted in suboptimal testing procedures worldwide. Alternative workflows have been implemented to overcome these difficulties. Recently a liquid based sample prep has been launched as solution to overcome limitations in relation to nucleic acid extraction.Objective: Multicenter evaluation of the QlAprep& Viral RNA UM kit (QIA P&A) for rapid sample preparation and real-time PCR detection of SARS-CoV-2 in comparison to standardized laboratory testing methods.Study design: Selected samples of the routine diagnostic workflow at Clinical Microbiology Laboratories of four Dutch hospitals have been subjected to the rapid QIA P&A protocol and the results have been compared to routine diagnostic data.Results: Combining results of manual and automated procedures, a total of 377 clinical samples of which 202 had been tested positive with a wide range of C-T values, showed almost complete concordance in the QIA P&A assay for samples up to C-T values of 33 with one exception of C-T 31. Prospectively 60 samples were tested and also showed 100 % concordance with 5 positives. The method has been automated by two centres.Conclusions: Despite an input of only 8 mu L of clinical sample, the QIA P&A kit showed good performance for sample preparation and amplification of SARS-CoV-2 and can contribute as a rapid molecular testing strategy in managing the CoV-2 pandemic. Show less
Metagenomic high-throughput sequencing (mHTS) is a hypothesis-free, universal pathogen detection technique for determination of the DNA/RNA sequences in a variety of sample types and infectious... Show moreMetagenomic high-throughput sequencing (mHTS) is a hypothesis-free, universal pathogen detection technique for determination of the DNA/RNA sequences in a variety of sample types and infectious syndromes. mHTS is still in its early stages of translating into clinical application. To support the development, implementation and standardization of mHTS procedures for virus diagnostics, the European Society for Clinical Virology (ESCV) Network on Next-Generation Sequencing (ENNGS) has been established. The aim of ENNGS is to bring together professionals involved in mHTS for viral diagnostics to share methodologies and experiences, and to develop application recommendations. This manuscript aims to provide practical recommendations for the wet lab procedures necessary for implementation of mHTS for virus diagnostics and to give recommendations for development and validation of laboratory methods, including mHTS quality assurance, control and quality assessment protocols. Show less
Ong, D.S.Y.; Claas, E.C.J.; Breijer, S.; Vaessen, N. 2020
Background: Due to the emergence of the coronavirus disease 2019 (COVID-19) pandemic there is an urgent need for rapid and accurate testing on the severe acute respiratory syndrome coronavirus 2 ... Show moreBackground: Due to the emergence of the coronavirus disease 2019 (COVID-19) pandemic there is an urgent need for rapid and accurate testing on the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).Objectives: The aim of this study was to assess the diagnostic performance of the GeneFinderTMCOVID-19 Plus RealAmp Kit on the ELITe InGenius sample-to-result platform, which is a commercial nucleic acid amplification test (NAT) targeting genes of SARS-CoV-2.Study design: Patients were eligible between March 18 and May 27, 2020, when they had respiratory symptoms that were suspected for COVID-19. The InGenius platform was compared to routine in-house NAT that was validated according to the national reference.Results: Of 128 randomly selected patients, 58 (45 %) tested positive and 55 (43 %) tested negative in both platforms. Sensitivity of the InGenius platform was 100 % (95 % confidence interval 94-100). In the remaining 15 (12 %) cases E and RdRp genes were not detected in both platforms but the nucleoprotein (N) gene was tested positive by the InGenius platform. All solitary N gene positive cases were confirmed by a N-gene specific in-house validated NAT, and most of these patients could also be considered positive based on other recently available COVID-19 positive respiratory samples or highly suspected radiological findings.Conclusion: The InGenius platform for SARS-CoV-2 detection has excellent sensitivity, is easy to use and provides fast results. The inclusion of the N gene as a third gene target may further increase sensitivity for the diagnosis of COVID-19 in comparison to the national reference method. Show less
Introduction: The SARS-CoV-2 pandemic of 2020 is a prime example of the omnipresent threat of emerging viruses that can infect humans. A protocol for the identification of novel coronaviruses by... Show moreIntroduction: The SARS-CoV-2 pandemic of 2020 is a prime example of the omnipresent threat of emerging viruses that can infect humans. A protocol for the identification of novel coronaviruses by viral metagenomic sequencing in diagnostic laboratories may contribute to pandemic preparedness.Aim: The aim of this study is to validate a metagenomic virus discovery protocol as a tool for coronavirus pandemic preparedness.Methods: The performance of a viral metagenomic protocol in a clinical setting for the identification of novel coronaviruses was tested using clinical samples containing SARS-CoV-2, SARS-CoV, and MERS-CoV, in combination with databases generated to contain only viruses of before the discovery dates of these coronaviruses, to mimic virus discovery.Results: Classification of NGS reads using Centrifuge and Genome Detective resulted in assignment of the reads to the closest relatives of the emerging coronaviruses. Low nucleotide and amino acid identity (81% and 84%, respectively, for SARS-CoV-2) in combination with up to 98% genome coverage were indicative for a related, novel coronavirus. Capture probes targeting vertebrate viruses, designed in 2015, enhanced both sequencing depth and coverage of the SARS-CoV-2 genome, the latter increasing from 71% to 98%.Conclusion: The model used for simulation of virus discovery enabled validation of the metagenomic sequencing protocol. The metagenomic protocol with virus probes designed before the pandemic, can assist the detection and identification of novel coronaviruses directly in clinical samples. Show less
Carbo, E.C.; Buddingh, E.P.; Karelioti, E.; Sidorov, I.A.; Feltkamp, M.C.W.; Borne, P.A. von dem; ... ; Vries, J.J.C. de 2020
Metagenomic sequencing is a powerful technique that enables detection of the full spectrum of pathogens present in any specimen in a single test. Hence, metagenomics is increasingly being applied... Show moreMetagenomic sequencing is a powerful technique that enables detection of the full spectrum of pathogens present in any specimen in a single test. Hence, metagenomics is increasingly being applied for detection of viruses in clinical cases with suspected infections of unknown etiology and a large number of relevant potential causes. This is typically the case in patients presenting with encephalitis, in particular when immunity is impaired by underlying disorders.In this study, viral metagenomics has been applied to a cohort of hematological patients with encephalitis of unknown origin.Because viral loads in cerebrospinal fluid of patients with encephalitis are generally low, the technical performance of a metagenomic sequencing protocol with viral enrichment by capture probes targeting all known vertebrate viral sequences was studied. Subsequently, the optimized viral metagenomics protocol was applied to a cohort of hematological patients with encephalitis of unknown origin.Viral enrichment by capture probes increased the viral sequence read count of metagenomics on cerebrospinal fluid samples 100 - 10.000 fold, compared to unenriched metagenomic sequencing.In five out of 41 (12%) hematological patients with encephalitis, a virus was detected by viral metagenomics which had not been detected by current routine diagnostics. BK polyomavirus, hepatitis E virus, human herpes virus-6 and Epstein Barr virus were identified by this unbiased metagenomic approach.This study demonstrated that hematological patients with encephalitis of unknown origin may benefit from early viral metagenomics testing as a single step approach. Show less
Background: Diagnosis of congenital viral infection at birth is generally attempted by direct detection of the virus by PCR in various neonatal materials. How to reliably diagnose intrauterine... Show moreBackground: Diagnosis of congenital viral infection at birth is generally attempted by direct detection of the virus by PCR in various neonatal materials. How to reliably diagnose intrauterine infection with parvovirus B19 (B19 V) at birth is unknown.Objectives: To evaluate the performance of B19 V DNA detection in cord blood (CB) or neonatal dried blood spots (DBS) in diagnosing fetal infection. Study design: Two cohorts of children diagnosed prenatally with an intrauterine B19 V infection were included in this study. CB samples of intrauterine B19 V infections that were sent to a reference laboratory for congenital infections in Stuttgart, Germany in the period 1995-2014 were tested in triplicate for B19 V DNA by quantitative PCR. DBS from children with intrauterine B19 V infection that underwent IUT at the LUMC, Leiden, the Netherlands in the period 2009-2014 were tested for B19 V DNA by quantitative B19 V PCR in triplicate.Results: Fourteen of twenty (70 %) CB samples tested positive for B19 V DNA. The positivity rate was 40 % (4/10) in those with a prenatal diagnosis< 20 weeks gestation. When intrauterine B19 V infection was diagnosed thereafter, 100 % (10/10) samples were B19 V DNA positive. Of the thirteen available DBS, twelve (92 %) tested positive. Viral load in CB and DBS corresponded inversely with time from fetal diagnosis to birth.Conclusion: B19 V DNA can be detected in neonatal blood samples of children following intrauterine B19 V infection, although the possibility of false-negatives, even in severe infections, should be considered. B19 V viral load at birth correlates with timing of infection. Show less
Wunderink, H.F.; Brouwer, C.S. de; Meijden, E. van der; Pastrana, D.V.; Kroes, A.C.M.; Buck, C.B.; Feltkamp, M.C.W. 2019
